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1.  Rapid identification and typing of Yersinia pestis and other Yersinia species by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry 
BMC Microbiology  2010;10:285.
Accurate identification is necessary to discriminate harmless environmental Yersinia species from the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis and from the group A bioterrorism plague agent Yersinia pestis. In order to circumvent the limitations of current phenotypic and PCR-based identification methods, we aimed to assess the usefulness of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) protein profiling for accurate and rapid identification of Yersinia species. As a first step, we built a database of 39 different Yersinia strains representing 12 different Yersinia species, including 13 Y. pestis isolates representative of the Antiqua, Medievalis and Orientalis biotypes. The organisms were deposited on the MALDI-TOF plate after appropriate ethanol-based inactivation, and a protein profile was obtained within 6 minutes for each of the Yersinia species.
When compared with a 3,025-profile database, every Yersinia species yielded a unique protein profile and was unambiguously identified. In the second step of analysis, environmental and clinical isolates of Y. pestis (n = 2) and Y. enterocolitica (n = 11) were compared to the database and correctly identified. In particular, Y. pestis was unambiguously identified at the species level, and MALDI-TOF was able to successfully differentiate the three biotypes.
These data indicate that MALDI-TOF can be used as a rapid and accurate first-line method for the identification of Yersinia isolates.
PMCID: PMC2992509  PMID: 21073689
2.  Detection of "Rickettsia sp. strain Uilenbergi" and "Rickettsia sp. strain Davousti" in Amblyomma tholloni ticks from elephants in Africa 
BMC Microbiology  2007;7:74.
To date, 6 tick-borne rickettsiae pathogenic for humans are known to occur in Africa and 4 of them were first identified in ticks before being recognized as human pathogens.
We examined 33 and 5 Amblyomma tholloni ticks from African elephants in the Central African Republic and Gabon, respectively, by PCR amplification and sequencing of a part of gltA and ompA genes of the genus Rickettsia. The partial sequences of gltA and ompA genes detected in tick in Gabon had 99.1% similarity with those of R. heilongjiangensis and 97.1% with those of Rickettsia sp. HL-93 strain, respectively. The partial gltA and ompA gene sequences detected in tick in the Central African Republic were 98.9% and 95.1% similar to those of Rickettsia sp. DnS14 strain and R. massiliae, respectively. Phylogenetic analysis showed Rickettsia sp. detected in Gabon clusters with R. japonica and R. heilongjiangensis in a phylogenetic tree based on the partial gltA and ompA genes. The genotype of the Rickettsia sp. detected in the Central African Republic is close to those of R. massiliae group in the phylogenetic tree based on partial gltA gene sequences, and distantly related to other rickettsiae in the tree based on partial ompA gene.
The degrees of similarity of partial gltA and ompA genes with recognized species indicate the rickettsiae detected in this study may be new species although we could only study the partial sequences of 2 genes regarding the amount of DNA that was available. We propose the Rickettsia sp. detected in Gabon be provisionally named "Rickettsia sp. stain Davousti" and Rickettsia sp. detected in the Central African Republic be named "Rickettsia sp. strain Uilenbergi".
PMCID: PMC1988807  PMID: 17683629
3.  Identification of rickettsial isolates at the species level using multi-spacer typing 
BMC Microbiology  2007;7:72.
In order to estimate whether multi-spacer typing (MST), based on the sequencing of variable intergenic spacers, could serve for the identification of Rickettsia at the species level, we applied it to 108 rickettsial isolates or arthropod amplicons that include representatives of 23 valid Rickettsia species.
MST combining the dksA-xerC, mppA-purC, and rpmE-tRNAfMet spacer sequences identified 61 genotypes, allowing the differentiation of each species by at least one distinct genotype. In addition, MST was discriminatory at the strain level in six species for which several isolates or arthropod amplicons were available.
MST proved to be a reproducible and high-resolution genotyping method allowing clear identification of rickettsial isolates at the species level and further additional differentiation of strains within some species.
PMCID: PMC1950309  PMID: 17662158
4.  Microarray for serotyping of Bartonella species 
BMC Microbiology  2007;7:59.
Bacteria of the genus Bartonella are responsible for a large variety of human and animal diseases. Serological typing of Bartonella is a method that can be used for differentiation and identification of Bartonella subspecies.
We have developed a novel multiple antigenic microarray to serotype Bartonella strains and to select poly and monoclonal antibodies. It was validated using mouse polyclonal antibodies against 29 Bartonella strains. We then tested the microarray for serotyping of Bartonella strains and defining the profile of monoclonal antibodies.
Bartonella strains gave a strong positive signal and all were correctly identified. Screening of monoclonal antibodies towards the Gro EL protein of B. clarridgeiae identified 3 groups of antibodies, which were observed with variable affinities against Bartonella strains.
We demonstrated that microarray of spotted bacteria can be a practical tool for serotyping of unidentified strains or species (and also for affinity determination) by polyclonal and monoclonal antibodies. This could be used in research and for identification of bacterial strains.
PMCID: PMC3225882  PMID: 17593301
5.  False positive PCR detection of Tropheryma whipplei in the saliva of healthy people 
BMC Microbiology  2007;7:48.
Tropheryma whipplei, the agent of Whipple's disease (WD), has been recently isolated and the genomes of two isolates have been fully sequenced. Previous diagnosis tools for the diagnosis of the disease used sequence analysis of the 16S rRNA gene. Using this target gene, the high percentage of detection of the bacterium in saliva of healthy people was in contrast to the negative results obtained with specific target genes. The aim of our study was to compare previously published primers targeting the 16S rRNA gene to real-time PCR with Taqman* probes targeting specific repeat genes only found in the genome of T. whipplei in a series of 57 saliva from healthy people.
Although the specific real-time PCR assays with both primers and probes were negative for all the samples, 13 out of 57 samples were positive with different primers previously reported targeting the 16S rRNA gene. Among the positive samples, 8 yielded a 231-bp sequence that was 99.1% identical to that of Actinomyces odontolyticus, 2 yielded a 226-bp that was 99.6% identical to that of A. turicensis, and 3 yielded a 160-bp sequence that was 98.5% identical to that of Capnocytophaga gingivalis. We found that the C. gingivalis and A. odontolyticus 16S rRNA sequences obtained in our study share more than 80% homology with the corresponding 16S rRNA sequences of the T. whipplei genomes especially at 5' and 3' end.
Asymptomatic carriers of T. whipplei in saliva may exist but their prevalence is much lower than those previously reported. Testing the specificity of designed primers is critical to avoid false positive detection of T. whipplei. In atypical case we recommend to test two different specific target genes before concluding.
PMCID: PMC1890548  PMID: 17535423
6.  SVARAP and aSVARAP: simple tools for quantitative analysis of nucleotide and amino acid variability and primer selection for clinical microbiology 
BMC Microbiology  2006;6:21.
Simple computerized methods that analyse variability along alignments of nucleotide or amino acid sequences can be very useful in a clinical microbiology laboratory for two main purposes. First, to optimize primer selection, which is critical for the identification of infectious pathogens based on gene sequencing: primers must target conserved nucleotide regions bordering highly variable areas to ensure discrimination of species. Second, it can be of interest to reveal mutations associated with drug resistance of pathogen agents. Our aim was therefore to test easy and cost-free tools (SVARAP and aSVARAP) that require short hands-on work, little expertise, and which allow visual interpretation and statistical analysis of results.
We first tested SVARAP to improve a strategy of identification of streptococci species of the Viridans Group targeting the groESL gene. Two regions with <500 nucleotides were identified, one being significantly more discriminant than one of a similar length used in a previous study (mean number of nucleotide differences between species, 113 (range: 12–193) vs. 77 (range: 14–109); p < 10-3). Secondly, aSVARAP was tested on reverse transcriptase (RT) sequences from 129 HIV-1 clinical strains to identify natural polymorphisms and drug-selected mutations emerging under nucleoside RT inhibitor (NRTI)-selective pressure. It revealed eleven of the 18 RT mutations considered in a reference HIV-1 genotypic NRTI-resistance interpretation algorithm.
SVARAP and aSVARAP are simple, versatile and helpful tools for analysis of sequence variability, and are currently being used in real practice in our clinical microbiology laboratory.
PMCID: PMC1453764  PMID: 16515699
7.  Sca1, a previously undescribed paralog from autotransporter protein-encoding genes in Rickettsia species 
BMC Microbiology  2006;6:12.
Among the 17 genes encoding autotransporter proteins of the "surface cell antigen" (sca) family in the currently sequenced Rickettsia genomes, ompA, sca5 (ompB) and sca4 (gene D), have been extensively used for identification and phylogenetic purposes for Rickettsia species. However, none of these genes is present in all 20 currently validated Rickettsia species. Of the remaining 14 sca genes, sca1 is the only gene to be present in all nine sequenced Rickettsia genomes. To estimate whether the sca1 gene is present in all Rickettsia species and its usefulness as an identification and phylogenetic tool, we searched for sca1genes in the four published Rickettsia genomes and amplified and sequenced this gene in the remaining 16 validated Rickettsia species.
Sca1 is the only one of the 17 rickettsial sca genes present in all 20 Rickettsia species. R. prowazekii and R. canadensis exhibit a split sca1 gene whereas the remaining species have a complete gene. Within the sca1 gene, we identified a 488-bp variable sequence fragment that can be amplified using a pair of conserved primers. Sequences of this fragment are specific for each Rickettsia species. The phylogenetic organization of Rickettsia species inferred from the comparison of sca1 sequences strengthens the classification based on the housekeeping gene gltA and is similar to those obtained from the analyses of ompA, sca5 and sca4, thus suggesting similar evolutionary constraints. We also observed that Sca1 protein sequences have evolved under a dual selection pressure: with the exception of typhus group rickettsiae, the amino-terminal part of the protein that encompasses the predicted passenger domain, has evolved under positive selection in rickettsiae. This suggests that the Sca1 protein interacts with the host. In contrast, the C-terminal portion containing the autotransporter domain has evolved under purifying selection. In addition, sca1 is transcribed in R. conorii, and might therefore be functional in this species.
The sca1 gene, encoding an autotransporter protein that evolves under dual evolution pressure, is the only sca-family gene to be conserved by all Rickettsia species. As such, it is a valuable identification target for these bacteria, especially because rickettsial isolates can be identified by amplification and sequencing of a discriminatory gene fragment using a single primer pair. It may also be used as a phylogenetic tool. However, its current functional status remains to be determined although it was found expressed in R. conorii.
PMCID: PMC1388218  PMID: 16504018
8.  Proposal to create subspecies of Rickettsia conorii based on multi-locus sequence typing and an emended description of Rickettsia conorii 
BMC Microbiology  2005;5:11.
Rickettsiae closely related to the Malish strain, the reference Rickettsia conorii strain, include Indian tick typhus rickettsia (ITTR), Israeli spotted fever rickettsia (ISFR), and Astrakhan fever rickettsia (AFR). Although closely related genotypically, they are distinct serotypically. Using multilocus sequence typing (MLST), we have recently found that distinct serotypes may not always represent distinct species within the Rickettsia genus. We investigated the possibility of classifying rickettsiae closely related to R. conorii as R. conorii subspecies as proposed by the ad hoc committee on reconciliation of approaches to bacterial systematics. For this, we first estimated their genotypic variability by using MLST including the sequencing of 5 genes, of 31 rickettsial isolates closely related to R. conorii strain Malish, 1 ITTR isolate, 2 isolates and 3 tick amplicons of AFR, and 2 ISFR isolates. Then, we selected a representative of each MLST genotype and used multi-spacer typing (MST) and mouse serotyping to estimate their degree of taxonomic relatedness.
Among the 39 isolates or tick amplicons studied, four MLST genotypes were identified: i) the Malish type; ii) the ITTR type; iii) the AFR type; and iv) the ISFR type. Among these four MLST genotypes, the pairwise similarity in nucleotide sequence varied from 99.8 to 100%, 99.4 to 100%, 98.2 to 99.8%, 98.4 to 99.8%, and 99.2 to 99.9% for 16S rDNA, gltA, ompA, ompB, and sca4 genes, respectively. Representatives of the 4 MLST types were also classified within four types using MST genotyping as well as mouse serotyping.
Although homogeneous genotypically, strains within the R. conorii species show MST genotypic, serotypic, and epidemio-clinical dissimilarities. We, therefore, propose to modify the nomenclature of the R. conorii species through the creation of subspecies. We propose the names R. conorii subsp. conorii subsp. nov. (type strain = Malish, ATCC VR-613), R. conorii subspecies indica subsp. nov. (type strain = ATCC VR-597), R. conorii subspecies caspia subsp. nov. (type strain = A-167), and R. conorii subspecies israelensis subsp. nov. (type strain = ISTT CDC1). The description of R. conorii is emended to accomodate the four subspecies.
PMCID: PMC1079849  PMID: 15766388
9.  Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism 
BMC Microbiology  2004;4:21.
The limited circulation of many of the agents that are likely to be used in a bioterrorism attack precludes the ready availability of positive controls. This means that only specialized laboratories can screen for the presence of these agents by nucleic amplification assays. Calibrated controls are also necessary for quantitative measurements. Primers and probes to be used in both conventional and real-time PCR assays were designed for the detection of agents likely to be used by a bioterrorist. Three plasmids, each of which contains 4 to 6 specific sequences from agents on the CDC Category A and B list (excluding RNA viruses) were constructed. Two plasmids incorporate the sequences of Category A and B agents, respectively. The third plasmid incorporates sequences from Variola major and organisms that cause rash-like illnesses that may be clinically confused with smallpox. An "exogenic sequence", introducing a NotI restriction site was incorporated in the native sequences of the bioterrorism agents inserted in plasmids. The designed molecular system for detection of bioterrorism agents was tested on each of these agents (except Monkeypox virus, Smallpox virus and 2 Burkholderia species for which no native DNA was available) and a collection of 50 isolates of C. burnetii using constructed plasmids as positive controls.
Designed primers and probes allowed molecular detection, in either single or multiplex assays, of agent-specific targets with analytical sensitivities of between 1 and 100 DNA copies. The plasmids could be used as positive controls. False-positive results due to contamination by the positive control were easily detected by sequencing and eliminated by digestion with NotI.
Plasmid A and B can be used as positive controls in molecular assays for the detection of bioterrorism agents in clinical specimens or environmental samples. Plasmid C can be used as a positive control in differentiation of vesicular rashes. It is also possible to avoid or to ensure immediate detection of false positive results due to contamination by positive controls using these plasmids. These plasmids and the corresponding primers and probes are immediately available for all clinical microbiology laboratories provided they have molecular amplification equipment.
PMCID: PMC425577  PMID: 15147587

Results 1-9 (9)